Regulated transcription of targeted genes and other biological events

Abstract

Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors, transcription factors, vesicle fusion proteins, and other classes of intra- and extracellular proteins. We have developed a general procedure for the regulated (inducible) dimerization or oligomerization of intracellular proteins. In principle, any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable, synthetic ligands. To illustrate the practice of tis invention, we have induced: (1) the intracellular aggregation of the cytoplasmic tail of the zeta chain of the T cell receptor (TCR)-CD3 complex thereby leading to signaling and transcription of a reporter gene, (2) the homodimerization of the cytoplasmic tail of the Fas receptor thereby leading to cell-specific apoptosis (programmed cell death) and (3) the heterodimerization of a DNA-binding domain (Gal4) and a transcription-activation domain (VP16) thereby leading to direct transcription of a reporter gene. Regulated intracellular protein association with our cell permeable, synthetic ligands offers new capabilities in biological research and medicine, in particular, in gene therapy. Using gene transfer techniques to introduce our artificial receptors, one can turn on or off the signaling pathways that lead to the overexpression of therapeutic proteins by administering orally active "dimerizers" or "de-dimerizers", respectively. Since cells from different recipients can be configured to have the pathway overexpress different therapeutic proteins for use in a variety of disorders, the dimerizers have the potential to serve as "universal drugs". They can also be viewed as cell permeable, organic replacements for therapeutic antisense agents or for proteins that would otherwise require intravenous injection or intracellular expression (e.g., the LDL receptor or the CFTR protein).

We claim: 
     
       1. A genetic construct encoding a chimeric protein which comprises: (a) a ligand-binding domain which binds to a selected ligand to form a ligand cross-linked protein complex including the chimeric protein, wherein the ligand has one or more of the following characteristics: (i) the ligand is not a protein,   (ii) the ligand has a molecular weight less than 5 kD, and   (iii) the ligand is membrane permeable; and     (b) an intracellular targeting domain which is heterologous with respect to the ligand binding domain and, as a result of formation of the ligand cross-linked protein complex, causes cellular localization of said ligand cross-linked protein complex.   
     
     
       2. The genetic construct according to claim 1, wherein the intracellular targeting domain comprises a secretory leader sequence, a membrane retention domain, a nuclear localization domain or a vesicle targeting domain. 
     
     
       3. The genetic construct according to claim 1, wherein the intracellular targeting domain is a membrane retention domain including a plasma membrane targeting sequence for attachment of a myristoyl moiety or prenyl moiety. 
     
     
       4. The genetic construct according to claim 1, wherein the intracellular targeting domain is a membrane retention domain including a transmembrane domain. 
     
     
       5. The genetic construct according to claim 1, wherein the intracellular targeting domain is a nuclear localization domain including a bipartite basic repeat for directing the chimeric protein to the nucleus. 
     
     
       6. The genetic construct according to claim 1, wherein the ligand-binding domain comprises a ligand-binding domain of a steroid receptor. 
     
     
       7. The genetic construct according to claim 1, wherein the ligand-binding domain comprises an antibiotic binding domain. 
     
     
       8. The genetic construct according to claim 1, wherein the ligand-binding domain comprises a ligand-binding domain of a tetracycline receptor. 
     
     
       9. The genetic construct according to claim 1, wherein the ligand-binding domain comprises an antibody domain. 
     
     
       10. The genetic construct according to claim 1, wherein the ligand-binding domain comprises a ligand-binding domain of a cyclophilin receptor. 
     
     
       11. The genetic construct according to claim 1, wherein the ligand-binding domain comprises a ligand binding domain of an immunophilin. 
     
     
       12. The genetic construct according to claim 1, wherein the ligand-binding domain binds to FK506, FK520, rapamycin or a derivative of any of the foregoing. 
     
     
       13. The genetic construct according to claim 1, wherein the ligand-binding domain comprises an FK506 binding domain of a human FK506 Binding Protein (FKBP12) or a variant thereof in which one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 are replaced by other amino acid residues. 
     
     
       14. The genetic construct according to claim 1, wherein the chimeric protein includes two or more ligand-binding domains. 
     
     
       15. The genetic construct according to claim 1, wherein the chimeric protein includes two or more ligand-binding, domains having, different ligand binding specificities. 
     
     
       16. The genetic construct according to claim 1, wherein the ligand-binding domain is from 50 to 350 amino acid residues in length. 
     
     
       17. The genetic construct according to claim 1, wherein the ligand-binding domains comprises a naturally-occurring peptide sequence. 
     
     
       18. The genetic construct according to claim 1, wherein the ligand-binding domain comprises a non-naturally-occurring peptide sequence. 
     
     
       19. The genetic construct according to claim 1, wherein the chimeric protein further comprises an action domain which induces a biological process. 
     
     
       20. A vector comprising the genetic construct according to claim 1, and further comprising one or more of (i) an origin of replication, (ii) a selectable marker, (iii) an amplifiable marker, and (iv) a transcriptional regulatory sequence for expressing the chimeric protein in mammalian cells. 
     
     
       21. The genetic construct according to claim 11, wherein the ligand-binding domain comprises a ligand-binding domain of an FK506 Binding Protein. 
     
     
       22. The genetic construct according to claim 11, wherein the ligand-binding domain is a ligand binding domain of a human FK506 Binding Protein. 
     
     
       23. The genetic construct according to claim 13, wherein one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 of the FKBP12 are replaced by amino acids independently selected from the group consisting of Val, Ala, Gly and Met. 
     
     
       24. The genetic construct according to claim 13, wherein one or both of Phe36 and Asp37 are replaced by amino acids independently selected from the group consisting of Val and Ala. 
     
     
       25. The genetic construct according to claim 13, wherein Phe36 is replaced by a valine residue. 
     
     
       26. The genetic construct according to claim 13, wherein Phe36 is replaced by a methionine residue. 
     
     
       27. The genetic construct according to claim 16, wherein the ligand-binding domain is from 50 to 200 amino acid residues in length. 
     
     
       28. The genetic construct according to claim 18, wherein the non-naturally-occurring ligand-binding domain differs from a naturally-occurring ligand binding domain by one or more amino acid residues, and binds a ligand which is not bound by the naturally-occurring ligand binding domain. 
     
     
       29. The genetic construct according to claim 19, wherein the action domain comprises: (a) a DNA binding domain;   (b) a transcriptional activation domain;   (c) a transcriptional repressor domain; or   (d) a signaling domain which induces a detectable biological process.   
     
     
       30. The genetic construct according to claim 19, wherein the biological process includes a detectable protein kinase activity. 
     
     
       31. The genetic construct according to claim 19, wherein the biological process includes a detectable phosphatase activity. 
     
     
       32. The genetic construct according to claim 19, wherein the biological process includes a detectable activity comprising reductase activity, cyclooxygenase activity or protease activity. 
     
     
       33. The genetic construct according to claim 19, wherein the biological process is selected from the group consisting of channel opening, ion release, acylation, methylation, hydrolysis, phosphorylation, dephosphorylation, change in redox states, and rearrangement reactions. 
     
     
       34. The genetic construct according to claim 19, wherein the action domain includes a cytoplasmic domain of a cell surface receptor, or a variant thereof sufficient to induce the biological process in the cell. 
     
     
       35. The genetic construct according to claim 19, wherein the biological process includes cell death. 
     
     
       36. The genetic construct according to claim 19, wherein the biological process includes regulation of gene transcription. 
     
     
       37. The vector according to claim 20, wherein the vector is a viral vector. 
     
     
       38. The genetic construct according to any of claims 2-11 or 21, wherein the chimeric protein further comprises an action domain which induces a biological process. 
     
     
       39. The genetic construct according to claim 34, wherein the action domain is derived from the cytoplasmic domain of a cell surface receptor selected from the group consisting of a tyrosine kinase receptor, a cytokine receptor and a growth factor receptor. 
     
     
       40. The genetic construct according to claim 34, wherein the receptor is selected from the group consisting of CD3ζ, CD3η, CD3γ, CD3δ, CD3ε, an interferon receptor, an interleukin receptor, a GM-CSF receptor, a LIF receptor, a CNTF receptor, an oncostatin M receptor, a TGF-β receptor, an EGF receptor, ATR2/neu, a HER2/neu, a HER3/c-erbB-3, Xmrk, an insulin receptor, an IGF-1 receptor, IRR, PDGF receptor, a CSF-1 receptor, c-kit, STK-1/flk-2, an FGF receptor, flg, bek, an NGF receptor, Ig-alpha/MB-1, Ig-beta/B29, Ror1 and Ror2. 
     
     
       41. The genetic construct according to claim 35, wherein the action domain comprises a cytoplasmic portion of a Fas or TNF receptor sufficient to induce cell death. 
     
     
       42. The genetic construct according to claim 36, wherein the biological process includes regulation of a gene having a transcriptional regulatory element selected from the group consisting of a cAMP responsive element, an SRE, a VL30, an RSRF, an ISRE, a GAS, an ARRE-1 and an ARRE-2. 
     
     
       43. The genetic construct according to claim 36, wherein the biological process includes regulation of expression of an endogenous gene of a cell in which the genetic construct is expressed. 
     
     
       44. The genetic construct according to claim 36, wherein the biological process includes regulation of expression of a heterologous gene included in a cell in which the genetic construct is expressed. 
     
     
       45. The vector according to claim 37, wherein the viral vector is an adenoviral vector. 
     
     
       46. The vector according to claim 37, wherein the viral vector is an adeno-associated viral vector. 
     
     
       47. The vector according to claim 37, wherein the viral vector is a Herpes simplex viral vector. 
     
     
       48. The vector according to claim 37, wherein the viral vector is a retroviral vector. 
     
     
       49. The genetic construct according to claim 38, wherein the chimeric protein includes an action domain comprising a cytoplasmic domain of a cell surface receptor, or a variant thereof sufficient to induce the biological process in the cell. 
     
     
       50. The genetic construct according to claim 38, wherein the biological process includes cell death. 
     
     
       51. The genetic construct according to claim 38, wherein the biological process includes regulation of gene transcription. 
     
     
       52. The genetic construct according to any of claims 1, 2, 13, 18, 21, 25, 28, 29, 34, 35, 36, 43, or 44, wherein the ligand-binding domain binds the ligand with a Kd less than or equal to 10 -6  M. 
     
     
       53. The genetic construct according to any of claims 1, 2, 13, 18, 21, 25, 28, 29, 34, 35, 36, 43 or 44, wherein the ligand-binding domain is provided in the chimeric protein as an intracellular domain. 
     
     
       54. The genetic construct according to any of claims 1, 2, 13, 18, 21, 25, 28, 29, 34, 35, 36, 43 or 44, wherein the ligand-binding domain comprises a ligand-binding domain of an intracellular protein. 
     
     
       55. The genetic construct according to any of claims 1, 2, 13, 18, 21, 25, 28, 29, 34, 35, 36, 43 or 44, wherein the ligand-binding domain binds a ligand which is a synthetic organic molecule having a molecular weight of less than 5 kD. 
     
     
       56. The genetic construct according to any of claims 1, 2, 13, 18, 21, 25, 28, 29, 34, 35, 36, 43 or 44, wherein the ligand-binding domain binds a ligand which is membrane permeable. 
     
     
       57. The genetic construct according to any of claims 1, 2, 13, 18, 21, 25, 28, 29, 34, 35, 36, 43 or 44, wherein the ligand-binding domain binds a ligand which is a macrocyclic compound. 
     
     
       58. The genetic construct according to any of claims 1-15, 18, 19, 23-26, 28-36 or 39-44, wherein the ligand cross-linked protein complex comprises two or more chimeric proteins. 
     
     
       59. The genetic construct according to claim 52 wherein the ligand-binding domain binds the ligand with a Kd less than or equal to 10 -8  M. 
     
     
       60. The genetic construct according to claim 52, wherein the ligand-binding domain is provided in the chimeric fused protein as an intracellular domain and binds a ligand which is membrane permeable. 
     
     
       61. The genetic construct according to claim 53, wherein the ligand-binding domain binds the ligand with a Kd less than or equal to 10 -6  M. 
     
     
       62. The genetic construct according to claim 57, wherein the macrocyclic compound comprises a macrolide. 
     
     
       63. The genetic construct according to claim 60, wherein the ligand-binding domain binds a ligand which is a synthetic organic molecule having a molecular weight of less than 5 kD. 
     
     
       64. The genetic construct according to claim 60, wherein the ligand cross-linked protein complex comprises two or more chimeric proteins. 
     
     
       65. A vector for transducing a host cell, which vector comprises (I) a coding sequence for a chimeric protein, the chimeric protein comprising: (a) a ligand-binding domain which binds to a selected ligand to form a ligand cross-linked protein complex including the chimeric protein, wherein the selected ligand has one or more of the following characteristics: (i) the ligand is not a protein,   (ii) the ligand has a molecular weight less than 5 kD, and   (iii) the ligand is membrane permeable; and     (b) an intracellular targeting domain which is heterologous with respect to the ligand binding domain and, as a result of formation of the ligand cross-linked protein complex, causes cellular localization of said ligand cross-linked protein complex; and     (II) a transcriptional regulatory sequence for expressing the chimeric protein in the host cell.   
     
     
       66. The vector according to claim 65, wherein the intracellular targeting domain comprises a secretory leader sequence, a membrane retention domain, a nuclear localization domain or a vesicle targeting domain. 
     
     
       67. The vector according to claim 65, wherein the intracellular targeting domain is a membrane retention domain including a plasma membrane targeting sequence for attachment of a myristoyl moiety or prenyl moiety. 
     
     
       68. The vector according to claim 65, wherein the intracellular targeting domain is a membrane retention domain including a transmembrane domain. 
     
     
       69. The vector according to claim 65, wherein the intracellular targeting domain is a nuclear localization domain including a bipartite basic repeat for directing the chimeric protein to the nucleus. 
     
     
       70. The vector according to claim 65, wherein the ligand-binding domain comprises a ligand-binding domain of a steroid receptor. 
     
     
       71. The vector according to claim 65, wherein the ligand-binding domain comprises an antibiotic binding domain. 
     
     
       72. The vector according to claim 65, wherein the ligand-binding domain comprises a ligand-binding domain of a tetracycline receptor. 
     
     
       73. The vector according to claim 65, wherein the ligand-binding domain comprises an antibody domain. 
     
     
       74. The vector according to claim 65, wherein the ligand-binding domain comprises a ligand-binding domain of a cyclophilin receptor. 
     
     
       75. The vector according to claim 65, wherein the ligand-binding domain comprises a ligand binding domain of an immunophilin. 
     
     
       76. The vector according to claim 65, wherein the ligand-binding domain binds to FK506, FK520, rapamycin or a derivative of any of the foregoing. 
     
     
       77. The vector according to claim 65, wherein the ligand-binding domain comprises an FK506 binding domain of a human FK506 Binding Protein (FKBP12) or a variant thereof in which one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 are replaced by other amino acid residues. 
     
     
       78. The vector according to claim 65, wherein the chimeric protein includes two or more ligand-binding domains. 
     
     
       79. The vector according to claim 65, wherein the chimeric protein includes two or more ligand-binding domains having different ligand binding specificities. 
     
     
       80. The vector according to claim 65, wherein the ligand-binding domain is from 50 to 350 amino acid residues in length. 
     
     
       81. The vector according to claim 65, wherein the ligand-binding domain comprises a naturally-occurring peptide sequence. 
     
     
       82. The vector according to claim 65, wherein the ligand-binding domain comprises a non-naturally-occurring peptide sequence. 
     
     
       83. The vector according to claim 65, wherein the chimeric protein further comprises an action domain which induces a biological process. 
     
     
       84. The vector according to claim 65, wherein the transcriptional regulatory sequence comprises a promoter for expressing the chimeric protein in a eucaryotic cell. 
     
     
       85. The vector according to claim 65, wherein the transcriptional regulatory sequence comprises a promoter for expressing the chimeric protein in a mammalian host cell. 
     
     
       86. The vector according to claim 65, wherein the vector is a viral vector. 
     
     
       87. The vector according to claim 65, further comprising one or more of the following: a bacterial or yeast origin of replication, a selectable marker, or an amplifiable marker. 
     
     
       88. The vector according to claim 75, wherein the ligand-binding domain comprises a ligand-binding domain of an FK506 Binding Protein. 
     
     
       89. The vector according to claim 75, wherein the ligand-binding domain is a ligand binding domain of a human FK506 Binding Protein. 
     
     
       90. The vector according to claim 77, wherein one or more of Tyr26, Phe36, Asp37, Tyr82 and Phe99 of the FKBP12 are replaced by amino acids independently selected from the group consisting of Val, Ala, Gly and Met. 
     
     
       91. The vector according to claim 77, wherein one or both of Phe36 and Asp37 are replaced by amino acids independently selected from the group consisting of Val and Ala. 
     
     
       92. The vector according to claim 77, wherein Phe36 is replaced by a valine residue. 
     
     
       93. The vector according to claim 77, wherein Phe36 is replaced by a methionine residue. 
     
     
       94. The vector according to claim 80, wherein the ligand-binding domain is from 50 to 200 amino acid residues in length. 
     
     
       95. The vector according to claim 82, wherein the non-naturally-occurring ligand-binding domain differs from a naturally-occurring ligand binding domain by one or more amino acid residues, and binds a ligand which is not bound by the naturally-occurring ligand binding domain. 
     
     
       96. The vector according to claim 83, wherein the action domain comprises: (a) a DNA binding domain;   (b) a transcriptional activation domain;   (c) a transcriptional repressor domain; or   (d) a signaling domain which induces a detectable biological process.   
     
     
       97. The vector according to claim 83, wherein the biological process includes a detectable activity comprising protein kinase activity, phosphatase activity, reductase activity, cyclooxygenase activity or protease activity. 
     
     
       98. The vector according to claim 83, wherein the biological process is selected from the group consisting of channel opening, ion release, acylation, methylation, hydrolysis, phosphorylation, dephosphorylation, change in redox states, and rearrangement reactions. 
     
     
       99. The vector according to claim 83, wherein the action domain includes a cytoplasmic domain of a cell surface receptor, or a variant thereof sufficient to induce the biological process in the cell. 
     
     
       100. The vector according to claim 83, wherein the action domain is derived from the cytoplasmic domain of a cell surface receptor selected from the group consisting of a tyrosine kinase receptor, a cytokine receptor and a growth factor receptor. 
     
     
       101. The vector according to claim 83, wherein the biological process includes cell death. 
     
     
       102. The vector according to claim 83, wherein the biological process includes regulation of gene transcription. 
     
     
       103. The vector according to claim 85, wherein the mammalian cell is a human cell. 
     
     
       104. The vector according to claim 85, wherein the mammalian cell is a cell type selected from the group consisting of muscle, hematopoietic, neural, mesenchymal, cutaneous, mucosal, stromal, spleen, reticuloendothelial, epithelial, endothelial, hepatic, kidney, gastrointestinal and pulmonary cells. 
     
     
       105. The vector according to claim 86, wherein the viral vector is an adenoviral vector, adeno-associated viral vector, Herpes simplex viral vector or retroviral vector. 
     
     
       106. The vector according to any of claims 66-79 or 89-93, wherein the chimeric protein further comprises an action domain which induces a biological process. 
     
     
       107. The vector according to claim 100, wherein the receptor is selected from the group consisting of CD3ζ, CD3η, CD3γ, CD3δ, CD3ε, an interferon receptor, an interleukin receptor, a GM-CSF receptor, a LIF receptor, a CNTF receptor, an oncostatin M receptor, a TGF-β receptor, an EGF receptor, ATR2/neu, a HER2/neu, a HER3/c-erbB-3, Xmrk, an insulin receptor, an IGF-1 receptor, IRK, PDGF receptor, a CSF-1 receptor, c-kit, STK-1/flk-2, an FGF receptor, flg, bek, an NGF receptor, Ig-alpha/MB-1, Ig-beta/B29, Ror1 and Ror2. 
     
     
       108. The vector according to claim 101, wherein the action domain comprises a cytoplasmic portion of a Fas or TNF receptor sufficient to induce cell death. 
     
     
       109. The vector according to claim 102, wherein the biological process includes regulation of a gene having a transcriptional regulatory element selected from the group consisting of a cAMP responsive element, an SRE, a VL30, an RSRF, an ISRE, a GAS, an ARRE-1 and an ARRE-2. 
     
     
       110. The vector according to claim 102, wherein the biological process includes regulation of expression of an endogenous gene of the cell. 
     
     
       111. The vector according to claim 102, wherein the biological process includes regulation of expression of a heterologous gene included in the cell. 
     
     
       112. The vector according to claim 106, wherein the chimeric protein includes an action domain comprising a cytoplasmic domain of a cell surface receptor, or a variant thereof sufficient to induce the biological process in the cell. 
     
     
       113. The vector according to claim 106, wherein the biological process includes cell death. 
     
     
       114. The genetic construct according to claim 106, wherein the biological process includes regulation of gene transcription. 
     
     
       115. The vector according to any of claims 65, 66, 77, 82, 88, 92, 95, 96, 101, 110 or 111, wherein the ligand-binding, domain binds the ligand with a Kd less than or equal to 10 -6  M. 
     
     
       116. The vector according to any of claims 65, 66, 77, 82, 88, 92, 95, 96, 101, 110 or 111, wherein the ligand-binding domain is provided in the chimeric protein as an intracellular domain. 
     
     
       117. The vector according to any of claims 65, 66, 77, 82, 88, 92, 95, 96, 101, 110 or 111, wherein the ligand-binding domain comprises a ligand-binding domain of an intracellular protein. 
     
     
       118. The vector according to any of claims 65, 66, 77, 82, 88, 92, 95, 96, 101, 110 or 111, wherein the ligand-binding domain binds a ligand which is a synthetic organic molecule having a molecular weight of less than 5 kD. 
     
     
       119. The vector according to any of claims 65, 66, 77, 82, 88, 92, 95, 96, 101, 110 or 111, wherein the ligand-binding domain binds a ligand which is membrane permeable. 
     
     
       120. The vector according to any of claims 65, 66, 77, 82, 88, 92, 95, 96, 101, 110 or 111, wherein the ligand-binding domain binds a ligand which is a macrocyclic compound. 
     
     
       121. The vector according to any of claims 65-79, 82, 83, 88-93, 95-102 or 107-111, wherein the ligand cross-linked protein complex comprises two or more chimeric proteins. 
     
     
       122. The vector according to claim 115, wherein the ligand-binding domain binds the ligand with a Kd less than or equal to 10 -8  M. 
     
     
       123. The vector according to claim 115, wherein the ligand-binding domain is provided in the chimeric protein as an intracellular domain and binds a ligand which is membrane permeable. 
     
     
       124. The vector according to claim 116, wherein the ligand-binding domain binds the ligand with a Kd less than or equal to 10 -6  M. 
     
     
       125. The vector according to claim 120, wherein the macrocyclic compound comprises a macrolide. 
     
     
       126. The vector according to claim 123, wherein the ligand-binding domain binds a ligand which is a synthetic organic molecule having a molecular weight of less than 5kD. 
     
     
       127. The vector according to claim 123, wherein the ligand cross-linked protein complex comprises two or more chimeric proteins.